CN118112112A - Method for detecting enantiomer in phenylephrine hydrochloride injection - Google Patents
Method for detecting enantiomer in phenylephrine hydrochloride injection Download PDFInfo
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- CN118112112A CN118112112A CN202311464611.7A CN202311464611A CN118112112A CN 118112112 A CN118112112 A CN 118112112A CN 202311464611 A CN202311464611 A CN 202311464611A CN 118112112 A CN118112112 A CN 118112112A
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- OCYSGIYOVXAGKQ-FVGYRXGTSA-N phenylephrine hydrochloride Chemical compound [H+].[Cl-].CNC[C@H](O)C1=CC=CC(O)=C1 OCYSGIYOVXAGKQ-FVGYRXGTSA-N 0.000 title claims abstract description 100
- 229960003733 phenylephrine hydrochloride Drugs 0.000 title claims abstract description 69
- 238000002347 injection Methods 0.000 title claims abstract description 30
- 239000007924 injection Substances 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 86
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 229960001802 phenylephrine Drugs 0.000 claims abstract description 7
- SONNWYBIRXJNDC-VIFPVBQESA-N phenylephrine Chemical compound CNC[C@H](O)C1=CC=CC(O)=C1 SONNWYBIRXJNDC-VIFPVBQESA-N 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 49
- 239000013558 reference substance Substances 0.000 claims description 37
- 238000000926 separation method Methods 0.000 claims description 27
- 239000011550 stock solution Substances 0.000 claims description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 19
- 229920000858 Cyclodextrin Polymers 0.000 claims description 19
- 239000001116 FEMA 4028 Substances 0.000 claims description 19
- 229960004853 betadex Drugs 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 17
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 16
- 238000007865 diluting Methods 0.000 claims description 16
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- 239000011259 mixed solution Substances 0.000 claims description 10
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 9
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 9
- 239000012488 sample solution Substances 0.000 claims description 8
- 239000012085 test solution Substances 0.000 claims description 8
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- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 3
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Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a method for detecting enantiomer in phenylephrine hydrochloride injection, which mainly comprises the steps of solution preparation, chromatographic conditions, detection of enantiomer in phenylephrine hydrochloride injection and the like. The test method can completely separate phenylephrine (S) -isomer from main components, thereby effectively controlling the quality of phenylephrine hydrochloride injection, more effectively realizing quality monitoring of phenylephrine hydrochloride injection and having practical guiding significance for improving the quality of products.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, relates to a method for detecting enantiomer in phenylephrine hydrochloride injection, and in particular relates to a method for measuring the accurate content of enantiomer in phenylephrine hydrochloride injection by using an HPLC method.
Background
Phenylephrine hydrochloride is an anti-shock vasoactive drug, is clinically used for treating toxic and anaphylactic shock and supraventricular tachycardia in infection, and is used for preventing and treating hypotension and mydriasis examination during general anesthesia and lumbar anesthesia, and the dosage form is injection. It can also be used for treating common cold, and relieving fever, headache, general soreness, nasal obstruction, watery nasal discharge, and sneeze caused by common cold or influenza. The structure of the phenylephrine hydrochloride contains a chiral carbon, so that phenylephrine hydrochloride enantiomer can be produced in the production process. Because phenylephrine hydrochloride has similar properties to enantiomers, the enantiomers are difficult to remove in the process of separating and purifying phenylephrine hydrochloride. This results in a decrease in the pharmacological activity of phenylephrine hydrochloride, and therefore the enantiomeric content of the product should be controlled to ensure the safety and effectiveness of clinical administration.
However, the detection method of phenylephrine hydrochloride injection is not clear in the current pharmacopoeias of various countries. According to the rule of the raw material import registration standard (JX 20150393), most of the raw materials adopt a normal-phase high-performance liquid chromatography chiral stationary phase method for enantiomer detection. However, because phenylephrine hydrochloride injection is an aqueous solution, and normal phase solvents have large harm, chiral chromatographic columns have high price and the like, the method for detecting the raw material medicine is not applicable. The reference literature of the currently known detection method of enantiomer in phenylephrine hydrochloride injection, namely high performance liquid chromatography chiral mobile phase method for determining the S-enantiomer content in phenylephrine hydrochloride injection, adopts a reversed-phase high performance liquid chromatography chiral mobile phase method and takes sulfobutyl-beta-cyclodextrin as a chiral additive.
However, the chiral additive sulfobutyl-beta-cyclodextrin has certain damage to the chromatographic column, and is easy to cause mechanical blockage of the chromatographic column, so that the column pressure is increased and the normal use cannot be realized. Therefore, how to control the content of enantiomer in phenylephrine hydrochloride injection and further ensure the quality of phenylephrine hydrochloride injection is important.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a method for detecting enantiomer in phenylephrine hydrochloride injection, which provides basis for quality standard formulation of phenylephrine hydrochloride injection so as to better control product quality and ensure safety and effectiveness of clinical medication.
The invention is mainly realized by the following technical scheme:
(1) Sample preparation:
preparing a test solution: diluting the product with water to obtain solution containing phenylephrine hydrochloride 0.1mg per 1 ml;
Preparing enantiomer reference stock solution: taking about 5mg of phenylephrine hydrochloride enantiomer reference substance, precisely weighing, placing into a 20ml measuring flask, adding water for dissolving and diluting to scale, and shaking uniformly;
preparing a reference substance solution: precisely measuring 1ml of enantiomer reference stock solution, placing into a100 ml measuring flask, diluting with water to scale, and shaking;
Preparing a system applicability solution: about 10mg of phenylephrine hydrochloride reference substance is taken and put into a 100ml measuring flask, a proper amount of water is added to dissolve, 1ml of reference substance solution is precisely added, water is used for dilution to scale, and shaking is carried out, thus preparing a mixed solution containing about 0.1mg phenylephrine hydrochloride and 2.5 mug phenylephrine hydrochloride enantiomer in each 1 ml;
(2) Setting chromatographic conditions:
Chromatographic column: agilent InfinityLab Poroshell 120A 120EC-C18 column, 150X 4.6mm,4 μm;
mobile phase: taking 10mmol/L sodium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B for isocratic elution;
column temperature: 30-40 ℃;
flow rate: 0.1-1.0 ml/min;
Sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm;
(3) And (3) detection:
Precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
According to an embodiment of the invention, the enantiomer is phenylephrine (S) -hydrochloride having a degree of separation from the main peak of not less than 1.5.
According to an embodiment of the invention, 5mmol/L sodium sulfobutyl-beta-cyclodextrin salt is added to the 10mmol/L sodium dihydrogen phosphate solution, and the pH is adjusted to 3.0 by phosphoric acid.
According to an embodiment of the invention, the volume ratio of mobile phase a to mobile phase B is 97:3 or 98:2 or 98.5:1.5.
According to an embodiment of the invention, the volume ratio of mobile phase a to mobile phase B is preferably 98:2.
According to an embodiment of the invention, the column temperature is 30 ℃.
According to an embodiment of the invention, the flow rate is 0.8ml/min.
According to an embodiment of the invention, the sample volume is 20 μl.
According to an embodiment of the invention, the wavelength is 215nm.
According to an embodiment of the present invention, the detection limit of the impurity is: the enantiomer detection limit concentration was 0.0151. Mu.g/ml, which corresponds to 0.015% of the concentration of the test sample.
The invention has the beneficial effects that:
The invention provides a method for detecting enantiomer in phenylephrine hydrochloride injection, which can completely separate phenylephrine hydrochloride (S) -isomer from main components, thereby effectively controlling the quality of phenylephrine hydrochloride injection, more effectively realizing quality monitoring on phenylephrine hydrochloride injection and having practical guiding significance for improving the quality of products. The separation and measurement method has the advantages of strong specificity, high accuracy, simple and convenient operation, and wide popularization and application values, and can create wider economic benefits.
Drawings
FIG. 1 is a system applicability solution chromatogram measured under the chromatographic conditions of example 1 of the present invention.
FIG. 2 is a system applicability solution chromatogram measured under the chromatographic conditions of example 2 of the present invention.
FIG. 3 is a graph of the system applicability solution chromatograms measured under different organic phase examples under the chromatographic conditions of example 3 of the present invention.
FIG. 4 is a chromatogram of a system applicability solution measured by adding 10mmol/L chiral additive under the chromatographic condition of example 4 of the present invention.
FIG. 5 is a chromatogram of a system applicability solution measured by adding 5mmol/L chiral additive under the chromatographic condition of example 4 of the present invention.
FIG. 6 is a system applicability solution chromatogram measured under the chromatographic conditions of example 5 of the present invention.
Detailed Description
The invention will now be described in further detail with reference to the following specific examples and figures, which are intended to be illustrative only and not limiting in any way, and the raw materials used, unless otherwise specified, may be either commercially available or self-made.
According to the invention, the optimal analysis conditions are optimized after a large number of experiments, chiral chromatography Agilent InfinityLab Poroshell EC-C18 columns are adopted, a mobile phase adopts sodium dihydrogen phosphate buffer salt-acetonitrile, the content of a chiral additive sulfobutyl-beta-cyclodextrin sodium salt is reduced by adjusting pH and strictly controlling the component proportion of the mobile phase, the damage to the chromatographic columns is reduced, the separation degree between a main peak and an (S) -isomer is improved, the separation degree is more than 1.5, and the separation effect is outstanding.
Example 1
The method in reference high performance liquid chromatography chiral mobile phase method for determining S-enantiomer content in phenylephrine hydrochloride injection is used for detecting (S) -enantiomer in phenylephrine hydrochloride injection.
(1) Instrument and chromatographic conditions: chromatographic column: welch Xtimate C18 (250 mm. Times.4.6 mm,3 μm); mobile phase a:10mmol/L sodium dihydrogen phosphate solution (10 mmol/L sodium sulfobutyl-beta-cyclodextrin, pH value is adjusted to 3.0 by phosphoric acid); mobile phase B: acetonitrile; isocratic elution, mobile phase a mobile phase b=98.5:1.5; flow rate: 0.8ml/min; detection wavelength: 215nm; column temperature: 30 ℃; sample injection amount: 10 μl.
(2) Preparing a test solution: the product is diluted with water to obtain a solution containing about 0.5mg of phenylephrine hydrochloride per 1 ml. 2 parts were prepared in parallel.
(3) Preparation of enantiomer control stock solution: about 1mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well.
(4) Preparing a reference substance solution: precisely measuring 1ml of the enantiomer reference stock solution prepared in the step (3), placing in a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
(5) Preparing a system applicability solution: taking about 10mg of phenylephrine hydrochloride reference substance, placing the phenylephrine hydrochloride reference substance into a 100ml measuring flask, adding a proper amount of water to dissolve, precisely adding 1ml of the reference substance solution prepared in the step (4), diluting with water to a scale, and shaking uniformly to prepare a mixed solution containing about 0.5mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1 ml.
(6) And (3) detection: precisely measuring 10 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 10 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
The detection result of phenylephrine hydrochloride enantiomer is shown in figure 1, when the concentration of a sample is 0.5mg/ml, the sample injection amount needs to be optimized to ensure the detection sensitivity because the concentration is too large and chromatographic peaks are split.
Example 2 (optimization of sample concentration)
(1) The apparatus and chromatographic conditions were the same as in example 1.
(2) Preparing a test solution: the product is diluted with water to prepare a solution containing about 0.1mg of phenylephrine hydrochloride per 1 ml. 2 parts were prepared in parallel.
(3) Preparation of enantiomer control stock solution: about 5mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well.
(4) Preparing a reference substance solution: precisely measuring 1ml of the enantiomer reference stock solution prepared in the step (3), placing in a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
(5) Preparing a system applicability solution: taking about 10mg of phenylephrine hydrochloride reference substance, placing the phenylephrine hydrochloride reference substance into a 100ml measuring flask, adding a proper amount of water to dissolve, precisely adding 1ml of the reference substance solution prepared in the step (4), diluting with water to a scale, and shaking uniformly to prepare a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1 ml.
(6) And (3) detection: precisely measuring 10 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 10 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
System applicability test phenylephrine hcl and enantiomer retention times and resolution results are shown in table 1:
TABLE 1
Under the measurement condition, the separation degree of phenylephrine hydrochloride and enantiomer peaks in the system applicability solution is more than 1.5, and the system applicability is good.
Comparative example 1 the sample concentration was adjusted to 0.1mg/ml. As a result of the test, as shown in FIG. 2, when the concentration of the sample was 0.1mg/ml, the degree of separation of the (S) -isomer from the main component was 3.0, and the degree of separation was satisfactory. However, the peak time is 34.834min, and the eluting strength is insufficient, so that the method needs to be optimized and the proper peak time is adjusted.
Example 3 (optimization of mobile phase ratio)
(1) Instrument and chromatographic conditions: chromatographic column: welch Xtimate C18 (250 mm. Times.4.6 mm,3 μm); mobile phase a:10mmol/L sodium dihydrogen phosphate solution (10 mmol/L sodium sulfobutyl-beta-cyclodextrin, pH value is adjusted to 3.0 by phosphoric acid); mobile phase B: acetonitrile; isocratic elution, mobile phase a mobile phase b=98:2 or 97:3; flow rate: 0.8ml/min; detection wavelength: 215nm; column temperature: 30 ℃; sample injection amount: 20 μl.
(2) Preparing a test solution: the product is diluted with water to prepare a solution containing about 0.1mg of phenylephrine hydrochloride per 1 ml. 2 parts were prepared in parallel.
(3) Preparation of enantiomer control stock solution: about 1mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well.
(4) Preparing a reference substance solution: precisely measuring 1ml of the enantiomer reference stock solution prepared in the step (3), placing in a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
(5) Preparing a system applicability solution: taking about 10mg of phenylephrine hydrochloride reference substance, placing the phenylephrine hydrochloride reference substance into a 100ml measuring flask, adding a proper amount of water to dissolve, precisely adding 1ml of the reference substance solution prepared in the step (4), diluting with water to a scale, and shaking uniformly to prepare a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1 ml.
(6) And (3) detection: precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
System applicability test phenylephrine hcl and enantiomer retention times and resolution results are shown in table 2:
TABLE 2
Under the measurement condition, the separation degree of phenylephrine hydrochloride and enantiomer peaks in the system applicability solution is more than 1.5, and the system applicability is good.
Comparative example 2, the mobile phase composition ratio was adjusted. As shown in fig. 3, the test results show that the separation degree is good when the organic phase ratio is increased to 98:2 under different organic phase ratios (97:3; 98:2; 98.5:1.5), the peak-out time is moderate, and the effect is not as good when the organic phase ratio is continuously increased to 98.5:1.5 as 98:2, so that the mobile phase a is selected to be the mobile phase b=98:2.
The addition of the sodium sulfobutyl-beta-cyclodextrin can realize chiral separation so as to obtain better separation effect, but the sodium sulfobutyl-beta-cyclodextrin has certain damage to the chromatographic column, so that the mechanical blockage of the chromatographic column is easy to cause the rise of the column pressure and the normal use of the chromatographic column is impossible. Thus, the concentration of the sodium salt of sulfobutyl- β -cyclodextrin should be minimized under conditions that are satisfactory for isolation.
Example 4 (reduction of chiral additive amount)
(1) Instrument and chromatographic conditions: chromatographic column: welch Xtimate C18 (250 mm. Times.4.6 mm,3 μm); mobile phase a:10mmol/L sodium dihydrogen phosphate solution (10 mmol/L or 5mmol/L sodium sulfobutyl-beta-cyclodextrin, pH value is adjusted to 3.0 by phosphoric acid); mobile phase B: acetonitrile; isocratic elution, mobile phase a mobile phase b=98:2; flow rate: 0.8ml/min; detection wavelength: 215nm; column temperature: 30 ℃; sample injection amount: 20 μl.
(2) Preparing a test solution: the product is diluted with water to prepare a solution containing about 0.1mg of phenylephrine hydrochloride per 1 ml. 2 parts were prepared in parallel.
(3) Preparation of enantiomer control stock solution: about 1mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well.
(4) Preparing a reference substance solution: precisely measuring 1ml of the enantiomer reference stock solution prepared in the step (3), placing in a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
(5) Preparing a system applicability solution: taking about 10mg of phenylephrine hydrochloride reference substance, placing the phenylephrine hydrochloride reference substance into a 100ml measuring flask, adding a proper amount of water to dissolve, precisely adding 1ml of the reference substance solution prepared in the step (4), diluting with water to a scale, and shaking uniformly to prepare a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1 ml.
(6) And (3) detection: precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
System applicability test phenylephrine hcl and enantiomer retention times and resolution results are shown in table 3:
TABLE 3 Table 3
Under the measurement condition, the separation degree of phenylephrine hydrochloride and enantiomer peaks in the system applicability solution is more than 1.5, and the system applicability is good.
Comparative example 3 the initial concentration of sodium sulfobutyl- β -cyclodextrin was 10mmol/L, and although a separation effect could be achieved (fig. 4:10mmol/L sodium sulfobutyl- β -cyclodextrin), sodium sulfobutyl- β -cyclodextrin was liable to cause damage to the column, thus reducing the concentration to 5mmol/L. As shown in FIG. 5 (5 mmol/L sodium sulfobutyl-beta-cyclodextrin), the concentration of sodium sulfobutyl-beta-cyclodextrin was reduced from 10mmol/L to 5mmol/L, (S) -isomer was well separated and the retention time was shortened. Therefore, the 5mmol/L sodium sulfobutyl-beta-cyclodextrin can not only meet the separation effect, but also reduce the damage to the chromatographic column.
Although the consumption of the sulfobutyl-beta-cyclodextrin sodium is reduced, the damage to the chromatographic column can be reduced, the selection of the chromatographic column can be optimized because the sulfobutyl-beta-cyclodextrin sodium is still added in the mobile phase, and the chromatographic column with larger particle size can be selected as much as possible.
Example 5 (selection of optimized column)
(1) Setting chromatographic conditions: chromatographic column: agilent InfinityLab Poroshell 120EC-C18 (4.6X105 mm,4 um); mobile phase a:10mmol/L sodium dihydrogen phosphate solution (5 mmol/L sodium sulfobutyl-beta-cyclodextrin, pH value is adjusted to 3.0 by phosphoric acid); mobile phase B: acetonitrile; isocratic elution, mobile phase a mobile phase b=98:2; flow rate: 0.8ml/min; detection wavelength: 215nm; column temperature: 30 ℃; sample injection amount: 20 μl.
(2) Preparing a test solution: the product is diluted with water to prepare a solution containing about 0.1mg of phenylephrine hydrochloride per 1 ml. 2 parts were prepared in parallel.
(3) Preparation of enantiomer control stock solution: about 1mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well.
(4) Preparing a reference substance solution: precisely measuring 1ml of the enantiomer reference stock solution prepared in the step (3), placing in a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
(5) Preparing a system applicability solution: taking about 10mg of phenylephrine hydrochloride reference substance, placing the phenylephrine hydrochloride reference substance into a 100ml measuring flask, adding a proper amount of water to dissolve, precisely adding 1ml of the reference substance solution prepared in the step (4), diluting with water to a scale, and shaking uniformly to prepare a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1 ml.
(6) And (3) detection: precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
System applicability test phenylephrine hcl and enantiomer retention times and resolution results are shown in table 4:
TABLE 4 Table 4
Under the measurement condition, the separation degree of phenylephrine hydrochloride and enantiomer peaks in the system applicability solution is more than 1.5, and the system applicability is good.
Comparative example 4, a chromatographic column with a larger particle size was selected. As shown in FIG. 6, the test results show that the (S) -isomer and the main peak form are better, the separation degree is good, and the blank solvent has no interference by using a chromatographic column Agilent InfinityLab Poroshell EC-C18 (4.6X105 mm,4 um), so that the method is suitable for detecting the (S) -isomer.
Test example 1 specificity
A diluent: water and its preparation method
System applicability solution preparation: about 10mg of phenylephrine hydrochloride reference substance is taken and put into a 100ml measuring flask, a proper amount of water is added to dissolve, 1ml of enantiomer reference substance solution is precisely added, the mixture is diluted to scale by water and uniformly shaken, and a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1ml is prepared.
And respectively injecting the diluent and the system applicability solution into a liquid chromatograph, and examining whether a blank solvent (diluent) has interference on a main peak and an enantiomer, and the separation condition of the enantiomer and the main peak.
Test example 2 linearity and Range
A diluent: water and its preparation method
A main component stock solution: taking a proper amount of phenylephrine hydrochloride reference substance, precisely weighing, adding a diluting agent, shaking to dissolve and quantitatively diluting to prepare a solution containing about 0.1mg of phenylephrine hydrochloride in each 1ml serving as a stock solution.
Linear solution: and diluting the main component stock solution with diluent to obtain solutions with different concentrations (quantitative limit, 20%, 30%, 40%, 100%, 200%).
And respectively taking the linear solutions with different concentrations, injecting the linear solutions into a liquid chromatograph, and examining the linear relation between the concentration and the peak response.
Test example 3 detection limit and quantitative limit
A diluent: water and its preparation method
A main component stock solution: taking a proper amount of phenylephrine hydrochloride reference substance, precisely weighing, adding a diluting agent, shaking to dissolve and quantitatively diluting to prepare a solution containing about 0.1mg of phenylephrine hydrochloride in each 1ml serving as a stock solution.
LOQ solution: and respectively taking a proper amount of enantiomer stock solution and main component stock solution, and diluting with a diluent to prepare a solution with a certain concentration until the signal to noise ratio of the enantiomer peak and the main peak is more than or equal to 10.
LOD solution: and respectively taking a proper amount of enantiomer stock solution and a proper amount of main component stock solution, and diluting with a diluent to prepare a solution with a certain concentration until the signal to noise ratio of an enantiomer peak and a main peak is more than or equal to 3.
Test example 4 precision
Preparing a diluent: water and its preparation method
System applicability solution preparation: about 10mg of phenylephrine hydrochloride reference substance is taken and put into a 100ml measuring flask, a proper amount of water is added to dissolve, 1ml of enantiomer reference substance solution is precisely added, the mixture is diluted to scale by water and uniformly shaken, and a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1ml is prepared.
Preparing a test solution: the product is diluted with water to prepare a solution containing about 0.1mg of phenylephrine hydrochloride per 1 ml.
Preparing a reference substance solution: about 1mg of phenylephrine hydrochloride enantiomer reference is taken, precisely weighed, placed in a 20ml measuring flask, dissolved by adding water, diluted to a scale and shaken well. Precisely measuring 1ml of the prepared enantiomer reference stock solution, placing into a 100ml measuring flask, adding water to dilute to scale, and shaking uniformly.
The solutions were injected into a liquid chromatograph, respectively, and the reproducibility of the method was examined.
Test example 5 solution stability
And (3) preparing the solution with the same precision as in test example 4, and respectively testing the stability of the sample solution and the control solution by using a liquid chromatograph at different room temperature.
Test example 6 durability
Preparing a diluent: water and its preparation method
System applicability solution preparation: about 10mg of phenylephrine hydrochloride reference substance is taken and put into a 100ml measuring flask, a proper amount of water is added to dissolve, 1ml of enantiomer reference substance solution is precisely added, the mixture is diluted to scale by water and uniformly shaken, and a mixed solution containing about 0.1mg of phenylephrine hydrochloride and 2.5 mug of phenylephrine hydrochloride enantiomer in each 1ml is prepared.
The separation of enantiomers from the main peaks was examined by taking the system-applicable solutions and slightly varying the flow rate of the chromatographic conditions, the column temperature and the organic phase ratio.
The detection method is verified in terms of specificity, linearity and range, detection limit and quantitative limit, precision, solution stability, durability and the like, and the verification result is shown in table 5.
TABLE 5
Claims (10)
1. A method for detecting enantiomer in phenylephrine hydrochloride injection is characterized by adopting high performance liquid chromatography for detection, and comprises the following specific steps:
(1) Sample preparation:
preparing a test solution: diluting the product with water to obtain solution containing phenylephrine hydrochloride 0.1mg per 1 ml;
Preparing enantiomer reference stock solution: taking about 5mg of phenylephrine hydrochloride enantiomer reference substance, precisely weighing, placing into a 20ml measuring flask, adding water for dissolving and diluting to scale, and shaking uniformly;
preparing a reference substance solution: precisely measuring 1ml of enantiomer reference stock solution, placing into a100 ml measuring flask, diluting with water to scale, and shaking;
Preparing a system applicability solution: about 10mg of phenylephrine hydrochloride reference substance is taken and put into a 100ml measuring flask, a proper amount of water is added to dissolve, 1ml of reference substance solution is precisely added, water is used for dilution to scale, and shaking is carried out, thus preparing a mixed solution containing about 0.1mg phenylephrine hydrochloride and 2.5 mug phenylephrine hydrochloride enantiomer in each 1 ml;
(2) Setting chromatographic conditions:
Chromatographic column: agilent InfinityLab Poroshell 120A 120EC-C18 column, 150X 4.6mm,4 μm; mobile phase: taking 10mmol/L sodium dihydrogen phosphate solution as a mobile phase A and acetonitrile as a mobile phase B for isocratic elution;
column temperature: 30-40 ℃;
flow rate: 0.1-1.0 ml/min;
Sample injection volume: 5-20 mul;
detection wavelength: 200-250 nm;
(3) And (3) detection:
Precisely measuring 20 mu 1 of system applicability solution, injecting into a liquid chromatograph, wherein the separation degree of phenylephrine hydrochloride enantiomer and main peak meets the requirement; then precisely measuring 20 mu 1 of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms.
2. The method according to claim 1, wherein the enantiomer is phenylephrine (S) -hydrochloride and the degree of separation from the main peak is not less than 1.5.
3. The method according to claim 1, wherein 5mmol/L sodium sulfobutyl- β -cyclodextrin is added to the 10mmol/L sodium dihydrogen phosphate solution, and the pH is adjusted to 3.0 with phosphoric acid.
4. The method according to claim 1, wherein the volume ratio of mobile phase a to mobile phase B is 97:3 or 98:2 or 98.5:1.5.
5. The method according to claim 4, wherein the volume ratio of mobile phase A to mobile phase B is preferably 98:2.
6. The method of claim 1, wherein the column temperature is 30 ℃.
7. The method of claim 1, wherein the flow rate is 0.8ml/min.
8. The method of claim 1, wherein the sample volume is 20 μl.
9. The method of claim 1, wherein the wavelength is 215nm.
10. The method of claim 1, wherein the impurity is detected at a limit of: the enantiomer detection limit concentration was 0.0151. Mu.g/ml, which corresponds to 0.015% of the concentration of the test sample.
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